Exercise induced stress in horses: Selection of the most stable reference genes for quantitative RT-PCR normalization

<p>Abstract</p> <p>Background</p> <p>Adequate stress response is a critical factor during athlete horses' training and is central to our capacity to obtain better performances while safeguarding animal welfare.</p> <p>In order to investigate the molecular mechanisms underlying this process, several studies have been conducted that take advantage of microarray and quantitative real-time PCR (qRT-PCR) technologies to analyse the expression of candidate genes involved in the cellular stress response.</p> <p>Appropriate application of qRT-PCR, however, requires the use of reference genes whose level of expression is not affected by the test, by general physiological conditions or by inter-individual variability.</p> <p>Results</p> <p>The expression of nine potential reference genes was evaluated in lymphocytes of ten endurance horses during strenuous exercise. These genes were tested by qRT-PCR and ranked according to the stability of their expression using three different methods (implemented in <it>geNorm</it>, <it>NormFinder </it>and <it>BestKeeper</it>). Succinate dehydrogenase complex subunit A (<it>SDHA</it>) and hypoxanthine phosphoribosyltransferase (<it>HPRT</it>) always ranked as the two most stably expressed genes. On the other hand, glyceraldehyde-3-phosphate dehydrogenase (<it>GAPDH</it>), transferrin receptor (<it>TFRC</it>) and ribosomal protein L32 (<it>RPL32</it>) were constantly classified as the less reliable controls.</p> <p>Conclusion</p> <p>This study underlines the importance of a careful selection of reference genes for qRT-PCR studies of exercise induced stress in horses. Our results, based on different algorithms and analytical procedures, clearly indicate <it>SDHA </it>and <it>HPRT </it>as the most stable reference genes of our pool.</p

Selection of reference genes for quantitative RT-PCR studies in striped dolphin (Stenella coeruleoalba) skin biopsies

BACKGROUND: Odontocete cetaceans occupy the top position of the marine food-web and are particularly sensitive to the bioaccumulation of lipophilic contaminants. The effects of environmental pollution on these species are highly debated and various ecotoxicological studies have addressed the impact of xenobiotic compounds on marine mammals, raising conservational concerns. Despite its sensitivity, quantitative real-time PCR (qRT-PCR) has never been used to quantify gene induction caused by exposure of cetaceans to contaminants. A limitation for the application of qRT-PCR is the need for appropriate reference genes which allow the correct quantification of gene expression. A systematic evaluation of potential reference genes in cetacean skin biopsies is presented, in order to validate future qRT-PCR studies aiming at using the expression of selected genes as non-lethal biomarkers. RESULTS: Ten commonly used housekeeping genes (HKGs) were partially sequenced in the striped dolphin (Stenella coeruleoalba) and, for each gene, PCR primer pairs were specifically designed and tested in qRT-PCR assays. The expression of these potential control genes was examined in 30 striped dolphin skin biopsy samples, obtained from specimens sampled in the north-western Mediterranean Sea. The stability of selected control genes was determined using three different specific VBA applets (geNorm, NormFinder and BestKeeper) which produce highly comparable results. Glyceraldehyde-3P-dehydrogenase (GAPDH) and tyrosine 3-monooxygenase (YWHAZ) always rank as the two most stably expressed HKGs according to the analysis with geNorm and Normfinder, and are defined as optimal control genes by BestKepeer. Ribosomal protein L4 (RPL4) and S18 (RPS18) also exhibit a remarkable stability of their expression levels. On the other hand, transferrin receptor (TFRC), phosphoglycerate kinase 1 (PGK1), hypoxanthine ribosyltransferase (HPRT1) and β-2-microglobin (B2M) show variable expression among the studied samples and appear as less suitable reference genes for data normalization. CONCLUSION: In this work, we have provided essential background information for the selection of control genes in qRT-PCR studies of cetacean skin biopsies, as a molecular technique to investigate ecotoxicological hazard in marine mammals. Of 10 HKGs tested, those encoding for YWHAZ and GAPDH appear as the most reliable control genes for the normalization of qRT-PCR data in the analysis of striped dolphin skin biopsies. Potentially useful reference genes are also those encoding for ribosomal proteins L4 and S18

Quantitative Real-Time PCR detection of TRPV1–4 gene expression in human leukocytes from healthy and hyposensitive subjects

<p>Abstract</p> <p>Background</p> <p>Besides functioning as chemosensors for a broad range of endogenous and synthetic ligands, transient receptor potential vanilloid (TRPV) 1–4 channels have also been related to capsaicin (TRPV1), pain, and thermal stimuli perception, and itching sensation (TRPV1–4). While the expression of the TRPV1–4 genes has been adequately proved in skin, sensory fibres and keratinocytes, less is known about TRPV3 and TRPV4 expression in human blood cells.</p> <p>Results</p> <p>To study the gene expression of TRPV1–4 genes in human leukocytes, a quantitative Real-Time PCR (qRT-PCR) method, based on the calculation of their relative expression, has been developed and validated. The four commonly used house-keeping genes (HKGs), β-Actin (Act-B), glyceraldehyde-3P-dehydrogenase (GAPDH), hypoxanthine ribosyltransferase (HPRT1), and cyclophilin B (hCyPB), were tested for the stability of their expression in several human leukocyte samples, and used in the normalization procedure to determine the mRNA levels of the TRPV 1–4 genes in 30 healthy subjects. cDNAs belonging to all the TRPV1–4 genes were detected in leukocytes but the genes appear to be expressed at different levels. Our analysis did not show significant sex differences in TRPV1–4 cDNA levels in the 30 healthy subjects. The same qRT-PCR assay was used to compare TRPV1–4 expression between healthy controls and patients hyposensitive to capsaicin, pain and thermal stimuli: an almost doubled up-regulation of the TRPV1 gene was found in the pathological subjects.</p> <p>Conclusion</p> <p>The qRT-PCR assay developed and tested in this study allowed us to determine the relative expression of TRPV1–4 genes in human leukocytes: TRPV3 is the least expressed gene of this pool, followed by TRPV4, TRPV1 and TRPV2. The comparison of TRPV1–4 gene expression between two groups of healthy and hyposensitive subjects highlighted the evident up-regulation of TRPV1, which was almost doubly expressed (1.9× normalized fold induction) in the latter group. All the four house-keeping genes tested in this work (Act-B, GAPDH, hCyPB, HPRT1) were classified as optimal controls and showed a constant expression in human leukocytes samples. We recommend the use of these genes in similar qRT-PCR studies on human blood cells.</p

Isolation of novel microsatellite loci in Orchesella villosa (Arthropoda, Collembola).

An experimental procedure using biotin-labelled probes and streptavidin-bound magnetic beads (FIASCO) was used to produce a microsatellite-enriched library for the collembolan Orchesella villosa. PCR primers were successfully constructed for seven loci containing, respectively, five pure, one interrupted, and one compound dinucleotide microsatellite repeats. As a preliminary test of their variability, we investigated 15 individuals from 5 locations inside a dismissed mining area in southern Tuscany. All microsatellite loci showed high levels of polymorphism. The mean number of different alleles at each locus across populations was 10.1 and observed heterozygosity per locus was 0.13–0.86. Only 2 out of the 7 loci appeared to be in Hardy–Weinberg equilibrium. The potential application of these loci to test the effects of environmental contamination on the genetic structure of exposed populations is discussed

The putative-farnesoic acid O-methyl transferase (FAMeT) gene of Ceratitis capitata: characterization and pre-imaginal life expression.

Farnesoic acid O-methyl transferase (FAMeT) is the enzyme involved in the penultimate step of insect juvenile hormone (JH) biosynthesis and is thus a key regulator in insect development and reproduction. We report the characterization of the putative-FAMeT in the medfly or Mediterranean fruit fly, Ceratitis capitata. This gene was identified by suppressive subtractive hybridization and completely sequenced by the screening of a medfly cDNA library. The obtained sequence was analyzed for conserved protein domain identification and its expression profile was evaluated by quantitative Real-Time PCR in medfly pre-imaginal life. The tissue expression of the isolated gene was verified by in situ hybridization on third instar larvae sections. The characterization of the isolated gene pointed out several typical features of methyl transferase genes. The pre-imaginal putative-FAMeT expression levels were consistent with JH titer change in Diptera. As recognized in some crustaceans, this gene seems to be widely expressed in the medfly as well. Ceratitis capitata is one of the most relevant agricultural pests against which insecticides and the sterile insect technique (SIT) are extensively used in spite of the well-known limitations of these approaches. Although results are not conclusive for the physiological role of the isolated gene, they suggest the characterization of a new gene in the Mediterranean fruit fly potentially involved in JH biosynthesis and may, therefore, have implications for pest control

Population genetics of three sympatric springtail species (Hexapoda: Collembola) from the South Shetland Islands: evidence for a common biogeographic pattern

Three sympatric springtail species, from the South Shetland Islands archipelago in the maritime Antarctic, are analysed here in a common biogeographic and evolutionary framework. This study was designed to compare their population genetic structure using the same molecular marker. Haplotype data for the mitochondrial cox1 gene have been obtained for seven populations of Folsomotoma octooculata and are compared with the data obtained, in previous studies and the current one, for the sympatric species Cryptopygus antarcticus antarcticus, and Friesea grisea. Molecular data obtained are consistent with the hypothesis that all species have been present in the archipelago since well before the last glacial maximum (around 20 000 ybp) and that their early diversifications appear to be linked with known interglacial periods in the region. These springtails may have survived the last glacial cycle in local refugia, from which they dispersed subsequently to ice-free ground re-exposed during the current interglacial period. The populations of the different species diversified at different times, although all of them are within the Pleistocene epoch. We propose that the earliest diversification of haplotypes in all three springtails in this archipelago occurred from local refugia in Livingston I., with subsequent spread of some haplotypes throughout the South Shetland Islands